Antisense oligonucleotide modulation of raf gene expression

ABSTRACT

Oligonucleotides are provided which are targeted to nucleic acids encoding human raf and capable of inhibiting raf expression. Methods of inhibiting the expression of human raf using oligonucleotides of the invention are also provided. The present invention further comprises methods of preventing or inhibiting hyperproliferation of cells and methods of treating abnormal proliferative conditions which employ oligonucleotides of the invention.

INTRODUCTION

This is a U.S. National Phase of PCT/US98/13961 filed Jul. 6, 1998,which is a continuation-in-part of U.S. patent application Ser. No.08/888,982 filed Jul. 7, 1997 issued as U.S. Pat. No. 5,981,731, whichis a continuation-in-part of U.S. patent application Ser. No. 08/756,806filed Nov. 26, 1996 issued as U.S. Pat. No. 5,952,229 which is acontinuation-in-part of PCT/US95/07111 filed May 31, 1995, which is acontinuation-in-part of U.S. patent application Ser. No. 08/250,856filed May 31, 1994 issued as U.S. Pat. No. 5,563,255. Each of theseapplications is assigned to the assignee of the present invention and isincorporated herein in its entirety.

FIELD OF THE INVENTION

This invention relates to compositions and methods for modulatingexpression of raf, a naturally present cellular protein which has beenimplicated in abnormal cell proliferation and tumor formation.Compositions and methods for modulating B-raf are provided. Thisinvention is also directed to methods for inhibiting hyperproliferationof cells; these methods can be used diagnostically or therapeutically.Furthermore, this invention is directed to treatment of conditionsassociated with expression of the raf gene.

BACKGROUND OF THE INVENTION

Alterations in the cellular genes which directly or indirectly controlcell growth and differentiation are considered to be the main cause ofcancer. The raf gene family includes three highly conserved genestermedA-, B- and c-raf (also called raf-1). Raf genes encode proteinkinases that are thought to play important regulatory roles in signaltransduction processes that regulate cell proliferation. Expression ofthe c-raf protein is believed to play a role in abnormal cellproliferation since it has been reported that 60% of all lung carcinomacell lines express unusually high levels of c-raf mRNA and protein. Rappet al., The Oncogene Handbook, E. P. Reddy, A. M Skalka and T. Curran,eds., Elsevier Science Publishers, New York, 1988, pp. 213-253. B-raf isstrongly activated by oncogenic ras. Marais et al., (1997) J. Biol.Chem. 272: 4378-4383.

Oligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. For example, workers inthe field have now identified antisense, triplex and otheroligonucleotide compositions which are capable of modulating expressionof genes implicated in viral, fungal and metabolic diseases.

Antisense oligonucleotides have been safely administered to humans andclinical trials of several antisense oligonucleotide drugs, targetedboth to viral and cellular gene products, are presently underway. Thephosphorothioate oligonucleotide, ISIS 2922, has been shown to beeffective against cytomegalovirus retinitis in AIDS patients. BioWorldToday, Apr. 29, 1994, p 3. The oligonucleotide drug, ISIS5132/CGP69846A,a phosphorothioate deoxyoligonucleotide targeted to human c-raf, iscurrently in Phase I clinical trials. This compound has shown potentantitumor activity in a variety of animal models of human tumors. It isthus established that oligonucleotides in general, and those targeted toraf in particular, can be useful therapeutic instrumentalities and canbe useful in treatment of cells and animal subjects, especially humans.

Antisense oligonucleotide inhibition of gene expression has proven to bea useful tool in understanding the roles of raf gene products. Anantisense oligonucleotide complementary to the first six codons of humanc-raf has been used to demonstrate that the mitogenic response of Tcells to interleukin-2 (IL-2) requires c-raf. Cells treated with theoligonucleotide showed a near-total loss of c-raf protein and asubstantial reduction in proliferative response to IL-2. Riedel et al.,Eur. J. Immunol. 1993, 23, 3146-3150. Rapp et al. have disclosedexpression vectors containing a raf gene in an antisense orientationdownstream of a promoter, and methods of inhibiting raf expression byexpressing an antisense Raf gene or a mutated Raf gene in a cell. WOapplication 93/04170. An antisense oligodeoxyribonucleotidecomplementary to codons 1-6 of murine c-Raf has been used to abolishinsulin stimulation of DNA synthesis in the rat hepatoma cell lineH4IIE. Tornkvist et al., J. Biol. Chem. 1994, 269, 13919-13921. WOApplication 93/06248 discloses methods for identifying an individual atincreased risk of developing cancer and for determining a prognosis andproper treatment of patients afflicted with cancer comprising amplifyinga region of the c-raf gene and analyzing it for evidence of mutation.

Denner et al. disclose antisense polynucleotides hybridizing to the genefor raf, and processes using them. WO 94/15645. Oligonucleotideshybridizing to human and rat raf sequences are disclosed.

Iversen et al. disclose heterotypic antisense oligonucleotidescomplementary to raf which are able to kill ras-activated cancer cells,and methods of killing raf-activated cancer cells. Numerousoligonucleotide sequences are disclosed, none of which are actuallyantisense oligonucleotide sequences. WO 94/23755.

SUMMARY OF THE INVENTION

The present invention provides oligonucleotides which are targeted tonucleic acids encoding human raf, particularly B-raf, and which arecapable of inhibiting raf expression. The oligonucleotides of theinvention are believed to be useful both diagnostically andtherapeutically, and are believed to be particularly useful in themethods of the present invention.

The present invention also comprises methods of inhibiting theexpression of human raf. These methods are believed to be useful boththerapeutically and diagnostically as a consequence of the associationbetween raf expression and hyperproliferation these methods are alsouseful as tools, for example for detecting and determining the role ofraf expression in various cell functions and physiological processes andconditions and for diagnosing conditions associated with raf expression.

The present invention also comprises methods of inhibitinghyperproliferation of cells using oligonucleotides of the invention.These methods are believed to be useful, for example in diagnosingraf-associated cell hyperproliferation. Methods of treating abnormalproliferative conditions are also provided. These methods employ theoligonucleotides of the invention. These methods are believed to beuseful both therapeutically and as clinical research and diagnostictools.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of bar graphs showing initial screens ofoligonucleotides targeted to human B-raf. FIG. 1A shows results foroligonucleotides ISIS 13720-13744. mRNA levels for both lower transcript(solid gray bars) and upper transcript (speckled bars) are shown. FIG.1B shows results for oligonucleotides 14132-14144, along with 13741,13742 and 13743. mRNA levels for both upper transcript (solid gray bars)and lower transcript (speckled bars) are shown.

FIG. 2 is a set of line graphs showing dose-response curves forreduction of B-raf mRNA levels in human A549 tumor cells byphosphorothioate oligodeoxynucleotides 13741 (open squares), 14144(diamonds), 14529 (circles), 14530 (triangles) and 14531 (crosshatchedsquares) after a 4-hour treatment in the presence of lipofectin. Resultsare normalized to G3PDH and expressed as a percent of control. FIG. 2Ashows a dose-response curve for inhibition of the upper B-raftranscript. FIG. 2B shows a dose-response curve for inhibition of thelower B-raf transcript.

FIG. 3 is a set of line graphs showing dose-response curves forreduction of B-raf mRNA levels in human T24 tumor cells byphosphorothioate oligodeoxynucleotides 13741 (open squares), 14144(diamonds), 14529 (circles), 14530 (triangles) and 14531 (crosshatchedsquares) after a 4-hour treatment in the presence of lipofectin. Resultsare normalized to G3PDH and expressed as a percent of control. FIG. 3Ashows a dose-response curve for inhibition of the upper B-raftranscript. FIG. 3B shows a dose-response curve for inhibition of thelower B-raf transcript.

FIG. 4 is a set of line graphs showing dose-response curves forreduction of B-raf mRNA levels in human T24 tumor cells byphosphorothioate oligodeoxynucleotides 13741 (squares) and2′-methoxyethoxy oligonucleotides 15341 (diamonds), 15342 (circles) and15344 (triangles) after a 4-hour treatment in the presence oflipofectin. Results are normalized to G3PDH and expressed as a percentof control. FIG. 4A shows a dose-response curve for inhibition of theupper B-raf transcript. FIG. 4B shows a dose-response curve forinhibition of the lower B-raf transcript.

DETAILED DESCRIPTION OF THE INVENTION

Malignant tumors develop through a series of stepwise, progressivechanges that lead to the loss of growth control characteristic of cancercells, i.e., continuous unregulated proliferation, the ability to invadesurrounding tissues, and the ability to metastasize to different organsites. Carefully controlled in vitro studies have helped define thefactors that characterize the growth of normal and neoplastic cells andhave led to the identification of specific proteins that control cellgrowth and differentiation. The raf genes are members of a gene familywhich encode related proteins termed A-, B- and c-raf. Raf genes codefor highly conserved serine-threonine-specific protein kinases which areknown to bind to the ras oncogene. They are part of a signaltransduction pathway believed to consist of receptor tyrosine kinases,p21 ras, Raf protein kinases, Mek1 (ERK activator or MAPKK) kinases andERK (MAPK) kinases, which ultimately phosphorylate transcriptionfactors. Signaling through this pathway can mediate differentiation,proliferation or oncogenic transformation in different cellularcontexts. Marais et al., (1997) J. Biol. Chem. 272: 4378-4383. Thus, rafkinases are believed to play a fundamental role in the normal cellularsignal transduction pathway, coupling a multitude of growth factors totheir net effect, cellular proliferation. Because rat proteins aredirect downstream effectors of ras protein function, therapies directedagainst raf kinases are believed to be useful in treatment ofras-dependent tumors. Monia et al. (1996) Nature Med. 2:668-675. The rafkinases are differentially regulated and expressed; c-raf, also known asraf-1, is the most thoroughly characterized and is expressed in allorgans and in all cell lines that have been examined. A- and B-raf arehighly expressed in urogenital and brain tissues, respectively. BecauseB-raf is highly expressed in neural tissues it was once thought to belimited to these tissues but it has since been found to be more widelyexpressed. Although all the raf kinases are bound by ras following rasstimulation, B-raf is most strongly activated (phosphorylated) byoncogenic ras, and may be the primary target of oncogenic ras in celltransformation. Marais et al., (1997) J. Biol. Chem. 272: 4378-4383.

Certain abnormal proliferative conditions are believed to be associatedwith raf expression and are, therefore, believed to be responsive toinhibition of raf expression. Abnormally high levels of expression ofthe raf protein are also implicated in transformation and abnormal cellproliferation. These abnormal proliferative conditions are also believedto be responsive to inhibition of raf expression. Examples of abnormalproliferative conditions are hyperproliferative disorders such ascancers, tumors, hyperplasias, pulmonary fibrosis, angiogenesis,psoriasis, atherosclerosis and smooth muscle cell proliferation in theblood vessels, such as stenosis or restenosis following angioplasty. Thecellular signalling pathway of which raf is a part has also beenimplicated in inflammatory disorders characterized by T-cellproliferation (T-cell activation and growth), such as tissue graftrejection, endotoxin shock, and glomerular nephritis, for example.

It has now been found that elimination or reduction of raf expressionmay halt or reverse abnormal cell proliferation. This has been foundeven when levels of raf expression are not abnormally high. There is agreat desire to provide compositions of matter which can modulate theexpression of raf. It is greatly desired to provide methods of detectionof nucleic acids encoding raf in cells, tissues and animals. It is alsodesired to provide methods of diagnosis and treatment of abnormalproliferative conditions associated with abnormal raf expression. Inaddition, kits and reagents for detection and study of nucleic acidsencoding raf are desired. “Abnormal” raf expression is defined herein asabnormally high levels of expression of the rat protein, expression ofan abnormal or mutant raf protein, or any level of raf expression in anabnormal proliferative condition or state.

The present invention employs oligonucleotides targeted to nucleic acidsencoding raf. This relationship between an oligonucleotide and itscomplementary nucleic acid target to which it hybridizes is commonlyreferred to as “antisense”. “Targeting” an oligonucleotide to a chosennucleic acid target, in the context of this invention, is a multistepprocess. The process usually begins with identifying a nucleic acidsequence whose function is to be modulated. This may be, as examples, acellular gene (or mRNA made from the gene) whose expression isassociated with a particular disease state, or a foreign nucleic acidfrom an infectious agent. In the present invention, the target is anucleic acid encoding raf; in other words, the raf gene or mRNAexpressed from the rat gene. The targeting process also includesdetermination of a site or sites within the nucleic acid sequence forthe oligonucleotide interaction to occur such that the desiredeffect—modulation of gene expression—will result. Once the target siteor sites have been identified, oligonucleotides are chosen which aresufficiently complementary to the target, i.e., hybridize sufficientlywell and with sufficient specificity, to give the desired modulation.

In the context of this invention “modulation” means either inhibition orstimulation. Inhibition of raf gene expression is presently thepreferred form of modulation. This modulation can be measured in wayswhich are routine in the art, for example by Northern blot assay of mRNAexpression or Western blot assay of protein expression as taught in theexamples of the instant application. Effects on cell proliferation ortumor cell growth can also be measured, as taught in the examples of theinstant application. “Hybridization”, in the context of this invention,means hydrogen bonding, also known as Watson-Crick base pairing, betweencomplementary bases, usually on opposite nucleic acid strands or tworegions of a nucleic acid strand. Guanine and cytosine are examples ofcomplementary bases which are known to form three hydrogen bonds betweenthem. Adenine and thymine are examples of complementary bases which formtwo hydrogen bonds between them. “Specifically hybridizable” and“complementary” are terms which are used to indicate a sufficient degreeof complementarity such that stable and specific binding occurs betweenthe DNA or RNA target and the oligonucleotide. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget interferes with the normal function of the target molecule tocause a loss of utility, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the oligonucleotide tonon-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment or, in the case of in vitro assays,under conditions in which the assays are conducted.

In preferred embodiments of this invention, oligonucleotides areprovided which are targeted to mRNA encoding B-raf. In accordance withthis invention, persons of ordinary skill in the art will understandthat mRNA includes not only the coding region which carries theinformation to encode a protein using the three letter genetic code,including the translation start and stop codons, but also associatedribonucleotides which form a region known to such persons as the5′-untranslated region, the 3′-untranslated region, the 5′ cap region,intron regions and intron/exon or splice junction ribonucleotides. Thus,oligonucleotides may be formulated in accordance with this inventionwhich are targeted wholly or in part to these associated ribonucleotidesas well as to the coding ribonucleotides. In preferred embodiments, theoligonucleotide is targeted to a translation initiation site (AUG codon)or sequences in the coding region, 5′ untranslated region or3′-untranslated region of the human B-raf mRNA. The functions ofmessenger RNA to be interfered with include all vital functions such astranslocation of the RNA to the site for protein translation, actualtranslation of protein from the RNA, splicing or maturation of the RNAand possibly even independent catalytic activity which may be engaged inby the RNA. The overall effect of such interference with the RNAfunction is to cause interference with raf protein expression.

The present invention provides oligonucleotides for modulation of rafgene expression. Such oligonucleotides are targeted to nucleic acidsencoding raf. Oligonucleotides and methods for modulation of B-raf areprovided; however, compositions and methods for modulating expression ofother forms of raf are also believed to have utility and arecomprehended by this invention. As hereinbefore defined, “modulation”means either inhibition or stimulation.

Inhibition of raf gene expression is presently the preferred form ofmodulation.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of nucleotide or nucleoside monomers consistingof naturally occurring bases, sugars and intersugar (backbone) linkages.The term “oligonucleotide” also includes oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake and increased stability in the presence ofnucleases.

Certain preferred oligonucleotides of this invention are chimericoligonucleotides. “Chimeric oligonucleotides” or “chimeras”, in thecontext of this invention, are oligonucleotides which contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionof modified nucleotides that confers one or more beneficial properties(such as, for example, increased nuclease resistance, increased uptakeinto cells, increased binding affinity for the RNA target) and a regionthat is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of antisense inhibition of gene expression.Consequently, comparable results can often be obtained with shorteroligonucleotides when chimeric oligos are used, compared tophosphorothioate deoxyoligonucleotides hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art. In one preferred embodiment, a chimericoligonucleotide comprises at least one region modified to increasetarget binding affinity, and, usually, a region that acts as a substratefor RNAse H. Affinity of an oligonucleotide for its target (in this casea nucleic acid encoding raf) is routinely determined by measuring the Tmof an oligonucleotide/target pair, which is the temperature at which theoligonucleotide and target dissociate; dissociation is detectedspectrophotometrically. The higher the Tm, the greater the affinity ofthe oligonucleotide for the target. In a more preferred embodiment, theregion of the oligonucleotide which is modified to increase raf mRNAbinding affinity comprises at least one nucleotide modified at the 2′position of the sugar, most preferably a 2′-O-alkyl, 2′-O-alkyl-O-alkylor 2′-fluoro-modified nucleotide. Such modifications are routinelyincorporated into oligonucleotides and these oligonucleotides have beenshown to have a higher Tm (i.e., higher target binding affinity) than2′-deoxyoligonucleotides against a given target. The effect of suchincreased affinity is to greatly enhance antisense oligonucleotideinhibition of raf gene expression. RNAse H is a cellular endonucleasethat cleaves the RNA strand of RNA:DNA duplexes; activation of thisenzyme therefore results in cleavage of the RNA target, and thus cangreatly enhance the efficiency of antisense inhibition. Cleavage of theRNA target can be routinely demonstrated by gel electrophoresis. Inanother preferred embodiment, the chimeric oligonucleotide is alsomodified to enhance nuclease resistance. Cells contain a variety of exo-and endo-nucleases which can degrade nucleic acids. A number ofnucleotide and nucleoside modifications have been shown to make theoligonucleotide into which they are incorporated more resistant tonuclease digestion than the native oligodeoxynucleotide. Nucleaseresistance is routinely measured by incubating oligonucleotides withcellular extracts or isolated nuclease solutions and measuring theextent of intact oligonucleotide remaining over time, usually by gelelectrophoresis. Oligonucleotides which have been modified to enhancetheir nuclease resistance survive intact for a longer time thanunmodified oligonucleotides. A variety of oligonucleotide modificationshave been demonstrated to enhance or confer nuclease resistance.Oligonucleotides which contain at least one phosphorothioatemodification are presently more preferred. In some cases,oligonucleotide modifications which enhance target binding affinity arealso, independently, able to enhance nuclease resistance. A discussionof antisense oligonucleotides and some desirable modifications can befound in De Mesmaeker et al., 1995, Acc. Chem. Res. 28:366-374.

Specific examples of some preferred oligonucleotides envisioned for thisinvention include those containing modified backbones, for example,phosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most preferred are oligonucleotideswith phosphorothioate backbones and those with heteroatom backbones,particularly CH₂—NH—O—CH₂, CH₂—N(CH₃)—O—CH₂ [known as amethylene(methylimino) or MMI backbone], CH₂—O—N(CH₃)—CH₂,CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH₂). The amidebackbones disclosed by De Mesmaeker et al. (1995, Acc. Chem. Res.28:366-374) are also preferred. Also preferred are oligonucleotideshaving morpholino backbone structures (Summerton and Weller, U.S. Pat.No. 5,034,506). In other preferred embodiments, such as the peptidenucleic acid (PNA) backbone, the phosphodiester backbone of theoligonucleotide is replaced with a polyamide backbone, the nucleobasesbeing bound directly or indirectly to the aza nitrogen atoms of thepolyamide backbone (Nielsen et al., Science, 1991, 254, 1497).Oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH, SH, SCH₃, F, OCN, OCH₃OCH₃, OCH₃O(CH₂)_(n)CH₃,O(CH₂)_(n)NH₂ or O(CH₂)_(n)CH₃ where n is from 1 to about 10; C₁ to C₁₀lower alkyl, alkoxyalkoxy (also known in the art as O-alkyl-O-alkyl),substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O—,S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂;heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino;substituted silyl; an RNA cleaving group; a reporter group; anintercalator; a group for improving the pharmacokinetic properties of anoligonucleotide; or a group for improving the pharmacodynamic propertiesof an oligonucleotide and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy [2′—O—CH₂CH₂OCH₃,also known as 2′—O—(2-methoxyethyl) or 2′-MOE] (Martin et al., Helv.Chim. Acta, 1995, 78, 486). Other preferred modifications include2′-methoxy (2′—O—CH₃), 2′-propoxy (2′—OCH₂CH₂CH₃) and 2′-fluoro (2′-F) .Similar modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide and the 5′ position of 5′ terminalnucleotide.Oligonucleotides may also have sugar mimetics such ascyclobutyls in place of the pentofuranosyl group.

Oligonucleotides may also include, additionally or alternatively,nucleobase (often referred to in the art simply as “base”) modificationsor substitutions. As used herein, “unmodified” or “natural” nucleobasesinclude adenine (A), guanine (G), thymine (T), cytosine (C) and uracil(U). Modified nucleobases include nucleobases found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2′deoxycytosine and often referred to in the artas 5-me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosylHMC, as well as synthetic nucleobases, e.g., 2-aminoadenine,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,8-azaguanine, 7-deazaguanine, N⁶(6-aminohexyl)adenine and2,6-diaminopurine. Kornberg, A., DNA Replication, W.H. Freeman & Co.,San Francisco, 1980, pp75-77; Gebeyehu, G., et al., 1987, Nucl. AcidsRes. 15:4513). A “universal” base known in the art, e.g., inosine, maybe included. 5-me-C substitutions have been shown to increase nucleicacid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke, S. T.and Lebleu, B., eds., Antisense Research and Applications, CRC Press,Boca Raton, 1993, pp. 276-278) and are presently preferred basesubstitutions.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety, a cholesteryl moiety (Letsingeret al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid(Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053), a thioether,e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), athiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259,327; Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, apolyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides& Nucleotides, 1995, 14, 969), or adamantane acetic acid (Manoharan etal., Tetrahedron Lett., 1995, 36, 3651). Oligonucleotides comprisinglipophilic moieties, and methods for preparing such oligonucleotides areknown in the art, for example, U.S. Pat. Nos. 5,138,045, 5,218,105 and5,459,255.

The oligonucleotides of the invention may be provided as prodrugs, whichcomprise one or more moieties which are cleaved off, generally in thebody, to yield an active oligonucleotide. One example of a prodrugapproach is described by Imbach et al. in WO Publication 94/26764.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even atwithin a single nucleoside within an oligonucleotide. The presentinvention also includes chimeric oligonucleotides as hereinbeforedefined.

The oligonucleotides in accordance with this invention preferably arefrom about 8 to about 50 nucleotides in length. In the context of thisinvention it is understood that this encompasses non-naturally occurringoligomers as hereinbefore described, having 8 to 50 monomers.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of the routineer. It is also well known to usesimilar techniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives. It is also well known touse similar techniques and commercially available modified amidites andcontrolled-pore glass (CPG) products such as biotin, fluorescein,acridine or psoralen-modified amidites and/or CPG (available from GlenResearch, Sterling Va.) to synthesize fluorescently labeled,biotinylated or other modified oligonucleotides such ascholesterol-modified oligonucleotides.

It has now been found that certain oligonucleotides targeted to portionsof the B-raf mRNA are useful for inhibiting raf expression. Inhibitionof B-raf expression using antisense oligonucleotides is believed to beuseful for interfering with cell hyperproliferation. In the methods ofthe invention, tissues or cells are contacted with oligonucleotides. Inthe context of this invention, to “contact” tissues or cells with anoligonucleotide or oligonucleotides means to add the oligonucleotide(s),usually in a liquid carrier, to a cell suspension or tissue sample,either in vitro or ex vivo, or to administer the oligonucleotide(s) tocells or tissues within an animal.

For therapeutics, methods of inhibiting hyperproliferation of cells andmethods of preventing and treating abnormal proliferative conditions areprovided. The formulation of therapeutic compositions and theirsubsequent administration is believed to be within the skill in the art.In general, for therapeutics, a patient suspected of needing suchtherapy is given an oligonucleotide in accordance with the invention,commonly in a pharmaceutically acceptable carrier, in amounts and forperiods which will vary depending upon the nature of the particulardisease, its severity and the patient's overall condition. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic, vaginal, rectal, intranasal,transdermal), oral or parenteral. Parenteral administration includesintravenous drip or infusion, subcutaneous, intraperitoneal orintramuscular injection, pulmonary administration, e.g., by inhalationor insufflation, or intrathecal or intraventricular administration. Fororal administration, it has been found that oligonucleotides with atleast one 2′-substituted ribonucleotide are particularly useful becauseof their absortion and distribution characteristics. U.S. Pat. No.5,591,721 (Agrawal et al.). Oligonucleotides with at least one2′-methoxyethyl modification are believed to be particularly useful fororal administration.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

Compositions for parenteral, intrathecal or intraventricularadministration may include sterile aqueous solutions which may alsocontain buffers, diluents and other suitable additives.

In addition to such pharmaceutical carriers, cationic lipids may beincluded in the formulation to facilitate oligonucleotide uptake. Onesuch composition shown to facilitate uptake is Lipofectin (BRL BethesdaMd.).

Dosing is dependent on severity and responsiveness of the condition tobe treated, with course of treatment lasting from several days toseveral months or until a cure is effected or a diminution of diseasestate is achieved. Optimal dosing schedules can be calculated frommeasurements of drug accumulation in the body. Persons of ordinary skillcan easily determine optimum dosages, dosing methodologies andrepetition rates. Optimum dosages may vary depending on the relativepotency of individual oligonucleotides, and can generally be calculatedbased on IC50's or EC50's in in vitro and in vivo animal studies. Forexample, given the molecular weight of compound (derived fromoligonucleotide sequence and chemical structure) and an effective dosesuch as an IC50, for example (derived experimentally), a dose in mg/kgis routinely calculated.

The present invention is also suitable for diagnosing abnormalproliferative states in tissue or other samples from patients suspectedof having a hyperproliferative disease such as cancer, psoriasis orblood vessel restenosis or atherosclerosis. The ability of theoligonucleotides of the present invention to inhibit cell proliferationmay be employed to diagnose such states. A number of assays may beformulated employing the present invention, which assays will commonlycomprise contacting a tissue sample with an oligonucleotide of theinvention under conditions selected to A permit detection and, usually,quantitation of such inhibition. Similarly, the present invention can beused to distinguish raf-associated, or, particularly, B-raf-associatedtumors from tumors having other etiologies, in order that an efficacioustreatment regime can be designed.

The oligonucleotides of this invention may also be used for researchpurposes. Thus, the specific hybridization exhibited by theoligonucleotides may be used for assays, purifications, cellular productpreparations and in other methodologies which may be appreciated bypersons of ordinary skill in the art.

The oligonucleotides of the invention are also useful for detection anddiagnosis of raf expression. For example, radiolabeled oligonucleotidescan be prepared by ³²p labeling at the 5′ end with polynucleotidekinase. Sambrook et al., Molecular Cloning. A Laboratory Manual, ColdSpring Harbor Laboratory Press, 1989, Volume 2, p. 10.59. Radiolabeledoligonucleotides are then contacted with tissue or cell samplessuspected of raf expression and the sample is washed to remove unboundoligonucleotide. Radioactivity remaining in the sample indicates boundoligonucleotide (which in turn indicates the presence of raf) and can bequantitated using a scintillation counter or other routine means.Radiolabeled oligo can also be used to perform autoradiography oftissues to determine the localization, distribution and quantitation ofraf expression for research, diagnostic or therapeutic purposes. In suchstudies, tissue sections are treated with radiolabeled oligonucleotideand washed as described above, then exposed to photographic emulsionaccording to routine autoradiography procedures. The emulsion, whendeveloped, yields an image of silver grains over the regions expressingraf. Quantitation of the silver grains permits raf expression to bedetected.

Analogous assays for fluorescent detection of raf expression can bedeveloped using oligonucleotides of the invention which are conjugatedwith fluorescein or other fluorescent tag instead of radiolabeling. Suchconjugations are routinely accomplished during solid phase synthesisusing fluorescently labeled amidites or CPG (e.g., fluorescein-labeledamidites and CPG available from Glen Research, Sterling Va. See 1993Catalog of Products for DNA Research, Glen Research, Sterling Va., p.21).

Each of these assay formats is known in the art. One of skill couldeasily adapt these known assays for detection of raf expression inaccordance with the teachings of the invention providing a novel anduseful means to detect raf expression.

Oligonucleotide Inhibition of B-raf Expression

The oligonucleotides shown in Table 1 were designed using the GenbankB-raf sequence HUMBRAF (SEQ ID NO: 42; Genbank listingsM95712;M95720;x54072), synthesized and tested for inhibition of B-rafmRNA expression in T24 bladder carcinoma cells or A549 lung carcinomacells using a Northern blot assay.

TABLE 1 Human B-raf Kinase Antisense Oligonucleotides (All arephosphorothioate oligodeoxynucleotides) Isis # Sequence (5′ → 3′) SiteSEQ ID NO: 13720 ATTTTGAAGGAGACGGACTG coding 1 13721TGGATTTTGAAGGAGACGGA coding 2 13722 CGTTAGTTAGTGAGCCAGGT coding 3 13723ATTTCTGTAAGGCTTTCACG coding 4 13724 CCCGTCTACCAAGTGTTTTC coding 5 13725AATCTCCCAATCATCACTCG coding 6 13726 TGCTGAGGTGTAGGTGCTGT coding 7 13727TGTAACTGCTGAGGTGTAGG coding 8 13728 TGTCGTGTTTTCCTGAGTAC coding 9 13729AGTTGTGGCTTTGTGGAATA coding 10 13730 ATGGAGATGGTGATACAAGC coding 1113731 GGATGATTGACTTGGCGTGT coding 12 13732 AGGTCTCTGTGGATGATTGA coding13 13733 ATTCTGATGACTTCTGGTGC coding 14 13734 GCTGTATGGATTTTTATCTTcoding 15 13735 TACAGAACAATCCCAAATGC coding 16 13736ATCCTCGTCCCACCATAAAA coding 17 13737 CTCTCATCTCTTTTCTTTTT coding 1813738 GTCTCTCATCTCTTTTCTTT coding 19 13739 CCGATTCAAGGAGGGTTCTG coding20 13740 TGGATGGGTGTTTTTGGAGA coding 21 13741 CTGCCTGGATGGGTGTTTTTcoding 22 14144 GGACAGGAAACGCACCATAT coding 23 14143CTCATTTGTTTCAGTGGACA stop codon 24 14142 TCTCTCACTCATTTGTTTCA stop codon25 14141 ACTCTCTCACTCATTTGTTT stop codon 26 14140 GAACTCTCTCACTCATTTGTcoding 27 14139 TCCTGAACTCTCTCACTCAT coding 28 14138TTGCTACTCTCCTGAACTCT 3′ UTR 29 14137 TTTGTTGCTACTCTCCTGAG coding 3014136 CTTTTGTTGCTACTCTCCTG 3′ UTR 31 13742 GCTACTCTCCTGAACTCTCT 3′ UTR32 14135 TTCCTTTTGTTGCTACTCTC 3′ UTR 33 14134 ATTTATTTTCCTTTTGTTGCcoding 34 14133 ATATGTTCATTTATTTTCCT coding 35 13743TTTATTTTCCTTTTGTTGCT 3′ UTR 36 13744 TGTTCATTTATTTTCCTTTT coding 3714132 ATTTAACATATAAGCAAACA coding 38 14529 CTGCCTGGTACCCTGTTTTT 5mismatch 39 14530 CTGCCTGGAAGGGTGTTTTT 1 mismatch 40 14531CTGCCTGGTACGGTGTTTTT 3 mismatch 41

There are multiple B-raf transcripts. The two most prevalent transcriptswere quantitated after oligonucleotide treatment. These transcripts runat approximately 8.5 kb (upper transcript) and 4.7 kb (lower transcript)under the gel conditions used. Both transcripts are translated intoB-raf protein in cells. In the initial screen, A549 cells were treatedwith oligonucleotides at a concentration of 200 nM oligonucleotide forfour hours in the presence of lipofectin. Results were normalized andexpressed as a percent of control. A graph showing oligonucleotideeffect on levels of B-raf mRNA (both upper and lower transcripts) isshown in FIG. 1 (panels A and B). In this initial screen,oligonucleotides giving a reduction of either B-raf mRNA transcript ofapproximately 30% or greater were considered active. According to thiscriterion, oligonucleotides 13722, 13724, 13726, 13727, 13728, 13730,13732, 13733, 13736, 13739, 13740, 13741, 13742, 13743, 14135, 14136,14138 and 14144 were found to be active. These sequences (SEQ ID NO: 3,5, 7, 8, 9, 11, 13, 14, 17, 20, 21, 22, 32, 36, 33, 31, 29 and 23,respectively) are therefore preferred. Of these, oligonucleotides 13727,13730, 13740, 13741, 13743 and 14144 (SEQ ID NO: 8, 11, 21, 22, 36 and23, respectively) showed 40-50% inhibition of one or both B-raftranscripts in at least one assay. These sequences are therefore morepreferred. In one of the two assays, ISIS 14144 (SEQ ID NO: 23) reducedlevels of both transcripts by 50-60% and ISIS 13741 (SEQ ID NO: 22)reduced both transcripts by 65-70%. These two sequences are thereforehighly preferred.

Dose response experiments were done in both T24 cells and A549 cells forthe two most active oligonucleotides, ISIS 13741 and ISIS 14144 (SEQ IDNO: 22 and 23), along with mismatch control sequences having 1, 3 or 5mismatches of the ISIS 13741 sequence (SEQ ID NO: 22). FIG. 2 showsdose-response curves for reduction of B-raf mRNA levels in A549 cells bythese oligonucleotides (all are phosphorothioate oligodeoxynucleotide5)after a 4-hour treatment in the presence of lipofectin. Results arenormalized to G3PDH and expressed as a percent of control. FIG. 2A showsa dose-response curve for inhibition of the upper B-raf transcript. ISIS13741 and 14144 had almost identical activity in this assay, with IC50sbetween 250 and 300 nM. The mismatch controls had no activity (ISIS145321) or slight activity, with a maximum inhibition of less than 20%at the 400 nM dose (ISIS 14530, ISIS 14529). FIG. 2B shows adose-response curve for inhibition of the lower B-raf transcript in A549cells. Against the lower transcript, ISIS 13741 and ISIS 14144 had IC50sof approximately 350 and 275 nM, respectively in this assay, with themismatch controls never achieving 50% inhibition at concentrations up to400 nM. Therefore, ISIS 13741 and 14144 are preferred.

FIG. 3 shows dose-response curves for reduction of B-raf mRNA levels inT24 cells by these oligonucleotides (all are phosphorothioateoligodeoxynucleotides) after a 4-hour treatment in the presence oflipofectin. Results are normalized to G3PDH and expressed as a percentof control. FIG. 3A shows a dose-response curve for inhibition of theupper B-raf transcript. ISIS 13741 and 14144 were again most active,with IC50s of approximately 100 nM and 275 nM, respectively, in thisassay. The mismatch controls 14529 and 14531 had no activity, and themismatch control 14530 achieved a maximum reduction of raf mRNA ofapproximately 20% at a 400 nM dose. FIG. 3B shows a dose-response curvefor inhibition of the lower B-raf transcript in T24 cells. Against thelower transcript, ISIS 13741 had an IC50 of approximately 100-125 nM andISIS 14144 had an IC50 of approximately 250 nM in this assay, with themismatch controls completely inactive. Therefore ISIS 13741 and 14144are preferred.

2′-Methoxyethoxy (2′-MOE) Oligonucleotides Targeted to B-raf

The oligonucleotides shown in Table 2 were synthesized. Nucleotidesshown in bold are 2′-MOE. 2′-MOE cytosines are all 5-methylcytosines.For backbone linkage, “s” indicates phosphorothioate (P═S) and “o”indicates phosphodiester (P=O).

TABLE 2 2′-MOE oligonucleotides targeted to human B-raf (bold = 2′-MOE)ISIS# Sequence/modification SEQ ID NO: 13741CsTsGsCsCsTsGsGsAsTsGsGsGsTsGsTsTsTsTsT 22 15339CsTsGsCsCsTsGsGsAsTsGsGsGsTsGsTsTsTsTsT 22 15340CoToGoCoCoToGoGoAoToGsGsGsTsGsTsTsTsTsT 22 15341CsTsGsCsCsTsGsGsAsTsGsGsGsTsGsTsTsTsTsT 22 15342CoToGoCoCsTsGsGsAsTsGsGsGsTsGoToToToToT 22 15343CsTsGsCsCsTsGsGsAsToGoGoGoToGoToToToToT 22 15344CsTsGsCsCsTsGsGsAsTsGsGsGsTsGsTsTsTsTsT 22

These oligonucleotides were tested for their ability to reduce B-rafmRNA levels in T24 cells. Dose response curves for ISIS 13741, 15341,15342 and 15344 are shown in FIG. 4A. FIG. 4A shows the effect of theseoligonucleotides on the lower B-raf transcript and FIG. 4B shows theeffect on the upper transcript. Against the lower transcript, ISIS 13741(P═S deoxy) and ISIS 15344 (P═S deoxy/MOE) had IC50s of approximately250 nM. The other two compound tested, ISIS 15341 and 15342, did notachieve 50% inhibition at doses up to 400 nM. Against the uppertranscript, ISIS 13741 and 15344 demonstrated IC50s of approximately 150nM, ISIS 15341 demonstrated an IC50 of approximately 200 nM and ISIS15342 did not achieve 50% reduction at doses up to 400 nM. Based onthese results, ISIS 15341, 13741 and 15344 are preferred. The followingexamples are provided for illustrative purposes only and are notintended to limit the invention.

EXAMPLES Example 1 Synthesis and Characterization of Oligonucleotides

Unmodified oligodeoxynucleotides are synthesized on an automated DNAsynthesizer (Applied Biosystems model 380B) using standardphosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl-phosphoramidites are purchased from AppliedBiosystems (Foster City, Calif.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2 M solution of³H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages. The thiation cycle wait step wasincreased to 68 seconds and was followed by the capping step.

2′-methoxy oligonucleotides were synthesized using 2′-methoxyβ-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham Mass.) andthe standard cycle for unmodified oligonucleotides, except the wait stepafter pulse delivery of tetrazole and base was increased to 360 seconds.Other 2′-alkoxy oligonucleotides were synthesized by a modification ofthis method, using appropriate 2′-modified amidites such as thoseavailable from Glen Research, Inc., Sterling, Va. 2′-fluorooligonucleotides were synthesized as described in Kawasaki et al., J.Med. Chem. 1993, 36, 831-841. Briefly, the protected nucleosideN⁶-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizingcommercially available 9-β-D-arabinofuranosyladenine as startingmaterial and by modifying literature procedures whereby the 2′-α-fluoroatom is introduced by a S_(N)2-displacement of a 2′-β-O-trifyl group.Thus N⁶-benzoyl-9-β-D-arabinofuranosyladenine was selectively protectedin moderate yield as the 3′, 5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N⁶-benzoyl groups was accomplished usingstandard methodologies and standard methods were used to obtain the5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished usingtetraisopropyldisiloxanyl (TPDS) protected 9-β-D-arabinofuranosylguanineas starting material, and conversion to the intermediatediisobutyrylarabinofuranosylguanosine. Deprotection of the TPDS groupwas followed by protection of the hydroxyl group with THP to givediisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-β-D-arabinofuranosyluracil was treated with 70% hydrogenfluoride-pyridine. Standard procedures were used to obtain the 5′-DMTand 5′-DMT-3′phosphoramidites. 2′-deoxy-2′-fluorocytidine wassynthesized via amination of 2′-deoxy-2′-fluorouridine, followed byselective protection to give N⁴-benzoyl-2′-deoxy-2′-fluorocytidine.Standard procedures were used to obtain the 5′-DMT and5′-DMT-3′phosphoramidites.

2′-(2-methoxyethyl)-modified amidites are synthesized according toMartin, P., Helv. Chim. Acta 1995, 78,486-504. For ease of synthesis,the last nucleotide was a deoxynucleotide. 2′—O—CH₂CH₂OCH₃-cytosines maybe 5-methyl cytosines.

Synthesis of 5-Methyl Cytosine Monomers

2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid which was crushed to a light tan powder (57 g, 85%crude yield). The material was used as is for further reactions.

2′-O-Methoxyethyl-5-methyluridine

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3)containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) andadsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5% Et₃NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by tlc by first quenching the tlc sample with the addition ofMeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 3 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the later solution. The resulting reaction mixture wasstored overnight in a cold room. Salts were filtered from the reactionmixture and the solution was evaporated. The residue was dissolved inEtOAc (1 L) and the insoluble solids were removed by filtration. Thefiltrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturatedNaCl, dried over sodium sulfate and evaporated. The residue wastriturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

A solution of3′-O-acetyl-2′-O-methoxyethyl1′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (tlc showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, tic showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/Hexane (1:1) containing 0.5% Et₃NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L).

Tetrazole diisopropylamine (7.1 g) and2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) were addedwith stirring, under a nitrogen atmosphere. The resulting mixture wasstirred for 20 hours at room temperature (tlc showed the reaction to be95% complete). The reaction mixture was extracted with saturated NaHCO₃(1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes wereback-extracted with CH₂Cl₂ (300 mL), and the extracts were combined,dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

5-methyl-2′-deoxycytidine (5-me-C) containing oligonucleotides weresynthesized according to published methods (Sanghvi et al., 1993, Nucl.Acids Res. 21:3197-3203) using commercially available phosphoramidites(Glen Research, Sterling Va. or ChemGenes, Needham Mass.).

Oligonucleotides having methylene(methylimino) backbones are synthesizedaccording to U.S. Pat. No. 5,378,825, which is coassigned to theassignee of the present invention and is incorporated herein in itsentirety. Other nitrogen-containing backbones are synthesized accordingto WO 92/20823 which is also coassigned to the assignee of the presentinvention and incorporated herein in its entirety.

Oligonucleotides having amide backbones are synthesized according to DeMesmaeker et al., Acc. Chem. Res. 1995, 28, 366-374. The amide moiety isreadily accessible by simple and well known synthetic methods and iscompatible with the conditions required for solid phase synthesis ofoligonucleotides.

Oligonucleotides with morpholino backbones are synthesized according toU.S. Pat. No. 5,034,506 (Summerton and Weller). Peptide-nucleic acid(PNA) oligomers are synthesized according to P. E. Nielsen et al.,Science 1991, 254, 1497).

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides are purified by precipitation twiceout of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotideswere analyzed by polyacrylamide gel electrophoresis on denaturing gelsand judged to be at least 85% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in synthesiswere periodically checked by ³¹P nuclear magnetic resonancespectroscopy, and for some studies oligonucleotides were purified byHPLC, as described by Chiang et al., 1991, J. Biol. Chem.,266:18162-18171. Results obtained with HPLC-purified material weresimilar to those obtained with non-HPLC purified material.

Example 2 Northern Blot Analysis of Inhibition of B-raf mRNA Expression

The human urinary bladder cancer cell line T24 and the human lung tumorcell line A549 were obtained from the American Type Culture Collection(Rockville Md.). T24 cells were grown in McCoy's 5A medium withL-glutamine and A549 cells were grown in DMEM low glucose medium (GibcoBRL, Gaithersburg Md.), supplemented with 10% heat-inactivated fetalcalf serum and 50 U/ml each of penicillin and streptomycin.

Cells were seeded on 100 mm plates. When they reached 70% confluency,they were treated with oligonucleotide. Plates were washed with 10 mlprewarmed PBS and 5 ml of Opti-MEM reduced-serum medium containing 2.5μl DOTMA per 100 nM oligonucleotide. Oligonucleotide with lipofectin wasthen added to the desired concentration. After 4 hours of treatment, themedium was replaced with appropriate medium (McCoy's or DMEM lowglucose). Cells were harvested 24 to 72 hours after oligonucleotidetreatment and RNA was isolated using a standard CsCl purificationmethod. Kingston, R. E., in Current Protocols in Molecular Biology, (F.M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G.Seidman and K. Strahl, eds.), John Wiley and Sons, N.Y. Total RNA wasisolated by centrifugation of cell lysates over a CsCl cushion. RNAsamples were electrophoresed through 1.2% agarose-formaldehyde gels andtransferred to hybridization membranes by capillary diffusion over a12-14 hour period. The RNA was cross-linked to the membrane by exposureto UV light in a Stratalinker (Stratagene, La Jolla, Calif.) andhybridized to a ³²P-labeled B-raf cDNA probe or G3PDH probe as acontrol. The human B-raf cDNA probe was cloned by PCR usingcomplementary oligonucleotide primers after reverse transcription oftotal RNA. Identity of the B-raf cDNA was confirmed by restrictiondigestion and direct DNA sequencing. RNA was quantitated using aPhosphorimager (Molecular Dynamics, Sunnyvale, Calif.).

Example 3 Antisense Inhibition of Cell Proliferation

T24 cells are treated on day 0 for two hours with various concentrationsof oligonucleotide and lipofectin (50 nM oligonucleotide in the presenceof 2 μg/ml lipofectin; 100 nM oligonucleotide and 2.5 μg/ml lipofectin;250 nM oligonucleotide and 6 μg/ml lipofectin or 500 nM oligonucleotideand 12.5 μg/ml lipofectin). On day 1, cells are treated for a secondtime at desired oligonucleotide concentration for two hours. On day 2,cells are counted.

Example 4 Effect of ISIS 13741 on T24 Human Bladder Carcinoma TumorXenografts in Nude Mice

5×10⁶ T24 cells are implanted subcutaneously in the right inner thigh ofnude mice. Oligonucleotides (ISIS 13741 and an unrelated controlphosphorothioate oligonucleotide suspended in saline) are administeredthree times weekly beginning on day 4 after tumor cell inoculation. Asaline-only control is also given. Oligonucleotides are given byintraperitoneal injection. Oligonucleotide dosage is 25 mg/kg. Tumorsize is measured and tumor volume is calculated on the eleventh,fifteenth and eighteenth treatment days.

Example 5 Effect of ISIS 13741 on MDA-MB 231 Human Breast CarcinomaTumor Xenografts in Nude Mice

5×10⁶ MDA-MB 231 cells are implanted subcutaneously in the right innerthigh of nude mice. Oligonucleotides (ISIS 13741 and an unrelatedcontrol phosphorothioate oligonucleotide suspended in saline) areadministered once daily beginning on day 10 after tumor cellinoculation. A saline-only control is also given. Oligonucleotides aregiven by intravenous injection at a dosage of 2-25 mg/kg. Tumor size ismeasured and tumor volume is calculated on days 10, 13, 16, 20, 23 and27 following tumor cell inoculation.

For intraperitoneal oligonucleotide administration, oligonucleotides areadministered once daily beginning on day 10 after tumor cellinoculation. A saline-only control is also given. Oligonucleotides aregiven by intraperitoneal injection at a dosage of 2-25 mg/kg. Tumor sizeis measured and tumor volume is calculated on days 10, 13, 16, 20, 23and 27 following tumor cell inoculation.

Example 6 Effect of ISIS 13741 on Colo 205 Human Colon Carcinoma TumorXenografts in Nude Mice

5×10⁶ Colo 205 cells are implanted subcutaneously in the right innerthigh of nude mice. Oligonucleotides (ISIS 13741 and an unrelatedcontrol phosphorothioate oligonucleotide suspended in saline) areadministered once per day beginning on day 5 after tumor cellinoculation. A saline-only control is also given. Oligonucleotides aregiven by intravenous injection. Oligonucleotide dosage is 2-25 mg/kg.Tumor size is measured and tumor volume is calculated on days 5, 8, 11,14, 18, 22 and after tumor inoculation.

Example 7 Diagnostic Assay for raf-associated Tumors Using Xenografts inNude Mice

Tumors arising from raf expression are diagnosed and distinguished fromother tumors using this assay. A biopsy sample of the tumor is treated,e.g., with collagenase or trypsin or other standard methods, todissociate the tumor mass. 5×10⁶ tumor cells are implantedsubcutaneously in the inner thighs of two or more nude mice. Antisenseoligonucleotide (e.g., ISIS 13741) suspended in saline is administeredto one or more mice by intraperitoneal injection three times weeklybeginning on day 4 after tumor cell inoculation. Saline only is given toa control mouse. Oligonucleotide dosage is 25 mg/kg. Tumor size ismeasured and tumor volume is calculated on the eleventh treatment day.Tumor volume of the oligonucleotide-treated mice is compared to that ofthe control mouse. The volume of raf-associated tumors in the treatedmice are measurably smaller than tumors in the control mouse. Tumorsarising from causes other than raf expression are not expected torespond to the oligonucleotides targeted to raf and, therefore, thetumor volumes of oligonucleotide-treated and control mice areequivalent.

Example 8 Detection of raf Expression

Oligonucleotides are radiolabeled after synthesis by ³²p labeling at the5′ end with polynucleotide kinase. Sambrook et al., Molecular Cloning. ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Volume 2,pg. 11.31-11.32. Radiolabeled oligonucleotides are contacted with tissueor cell samples suspected of raf expression, such as tumor biopsysamples, under conditions in which specific hybridization can occur, andthe sample is washed to remove unbound oligonucleotide. Radioactivityremaining in the sample indicates bound oligonucleotide and isquantitated using a scintillation counter or other routine means.

Radiolabeled oligonucleotides of the invention are also used inautoradiography. Tissue sections are treated with radiolabeledoligonucleotide and washed as described above, then exposed tophotographic emulsion according to standard autoradiography procedures.The emulsion, when developed, yields an image of silver grains over theregions expressing raf. The extent of raf expression is determined byquantitation of the silver grains.

Analogous assays for fluorescent detection of raf expression useoligonucleotides of the invention which are labeled with fluorescein orother fluorescent tags. Labeled DNA oligonucleotides are synthesized onan automated DNA synthesizer (Applied Biosystems model 380B) usingstandard phosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl phosphoramidites are purchased from AppliedBiosystems (Foster City, Calif.). Fluorescein-labeled amidites arepurchased from Glen Research (Sterling Va.). Incubation ofoligonucleotide and biological sample is carried out as described forradiolabeled oligonucleotides except that instead of a scintillationcounter, a fluorimeter or fluorescence microscope is used to detect thefluorescence which indicates raf expression.

Example 9 A549 Xenografts

A549 cells are obtained from the American Type Culture Collection(Bethesda Md.) and grown in T-75 flasks until 65-75% confluent. 5×10⁶A549 cells are implanted subcutaneously in the inner thigh of nude mice.Oligonucleotides (ISIS 13741 and a scrambled raf controlphosphorothioate oligonucleotide, ISIS 10353) suspended in saline areadministered once daily by intravenous injection at doses ranging from2-25 mg/kg. Resulting tumors are measured on days 9, 12, 17 and 21 andtumor volumes are calculated.

Example 10 U-87 Human Glioblastoma Cell Culture and SubcutaneousXenografts into Nude Mice

Ahmad et al. disclose that transfection of the human glioblastoma cellline, U-87, with vectors expressing antisense RNA to PKCα inhibitsgrowth of the glioblastoma cells in vitro and in vivo. Ahmad et al.,1994, Neurosurg. 35:904-908. The U-87 human glioblastoma cell line isobtained from the ATCC (Rockville Md.) and maintained in Iscove's DMEMmedium supplemented with heat-inactivated 10% fetal calf serum. Nudemice are injected subcutaneously with 2×10⁷ cells. Mice are injectedintraperitoneally with oligonucleotide at dosages of either 2 mg/kg or20 mg/kg for 21 consecutive days beginning 7 days after xenografts wereimplanted. Tumor volumes are measured on days 14, 21, 24, 31 and 35 andcompared to volumes obtained after treatment with saline or senseoligonucleotide control.

Example 11 Intracerebral U-87 Glioblastoma Xenografts in Nude Mice

U-87 cells are implanted in the brains of nude mice. Mice were treatedvia continuous intraperitoneal administration of antisenseoligonucleotide (20 mg/kg), control sense oligonucleotide (20 mg/kg) orsaline beginning on day 7 after xenograft implantation. Survival (indays) of treated mice are compared to survival of untreated orcontrol-treated mice.

42 20 Nucleic Acid Single Linear Yes 1 ATTTTGAAGG AGACGGACTG 20 20Nucleic Acid Single Linear Yes 2 TGGATTTTGA AGGAGACGGA 20 20 NucleicAcid Single Linear Yes 3 CGTTAGTTAG TGAGCCAGGT 20 20 Nucleic Acid SingleLinear Yes 4 ATTTCTGTAA GGCTTTCACG 20 20 Nucleic Acid Single Linear Yes5 CCCGTCTACC AAGTGTTTTC 20 20 Nucleic Acid Single Linear Yes 6AATCTCCCAA TCATCACTCG 20 20 Nucleic Acid Single Linear Yes 7 TGCTGAGGTGTAGGTGCTGT 20 20 Nucleic Acid Single Linear Yes 8 TGTAACTGCT GAGGTGTAGG20 20 Nucleic Acid Single Linear Yes 9 TGTCGTGTTT TCCTGAGTAC 20 20Nucleic Acid Single Linear Yes 10 AGTTGTGGCT TTGTGGAATA 20 20 NucleicAcid Single Linear Yes 11 ATGGAGATGG TGATACAAGC 20 20 Nucleic AcidSingle Linear Yes 12 GGATGATTGA CTTGGCGTGT 20 20 Nucleic Acid SingleLinear Yes 13 AGGTCTCTGT GGATGATTGA 20 20 Nucleic Acid Single Linear Yes14 ATTCTGATGA CTTCTGGTGC 20 20 Nucleic Acid Single Linear Yes 15GCTGTATGGA TTTTTATCTT 20 20 Nucleic Acid Single Linear Yes 16 TACAGAACAATCCCAAATGC 20 20 Nucleic Acid Single Linear Yes 17 ATCCTCGTCC CACCATAAAA20 20 Nucleic Acid Single Linear Yes 18 CTCTCATCTC TTTTCTTTTT 20 20Nucleic Acid Single Linear Yes 19 GTCTCTCATC TCTTTTCTTT 20 20 NucleicAcid Single Linear Yes 20 CCGATTCAAG GAGGGTTCTG 20 20 Nucleic AcidSingle Linear Yes 21 TGGATGGGTG TTTTTGGAGA 20 20 Nucleic Acid SingleLinear Yes 22 CTGCCTGGAT GGGTGTTTTT 20 20 Nucleic Acid Single Linear Yes23 GGACAGGAAA CGCACCATAT 20 20 Nucleic Acid Single Linear Yes 24CTCATTTGTT TCAGTGGACA 20 20 Nucleic Acid Single Linear Yes 25 TCTCTCACTCATTTGTTTCA 20 20 Nucleic Acid Single Linear Yes 26 ACTCTCTCAC TCATTTGTTT20 20 Nucleic Acid Single Linear Yes 27 GAACTCTCTC ACTCATTTGT 20 20Nucleic Acid Single Linear Yes 28 TCCTGAACTC TCTCACTCAT 20 20 NucleicAcid Single Linear Yes 29 TTGCTACTCT CCTGAACTCT 20 20 Nucleic AcidSingle Linear Yes 30 TTTGTTGCTA CTCTCCTGAG 20 20 Nucleic Acid SingleLinear Yes 31 CTTTTGTTGC TACTCTCCTG 20 20 Nucleic Acid Single Linear Yes32 GCTACTCTCC TGAACTCTCT 20 20 Nucleic Acid Single Linear Yes 33TTCCTTTTGT TGCTACTCTC 20 20 Nucleic Acid Single Linear Yes 34 ATTTATTTTCCTTTTGTTGC 20 20 Nucleic Acid Single Linear Yes 35 ATATGTTCAT TTATTTTCCT20 20 Nucleic Acid Single Linear Yes 36 TTTATTTTCC TTTTGTTGCT 20 20Nucleic Acid Single Linear Yes 37 TGTTCATTTA TTTTCCTTTT 20 20 NucleicAcid Single Linear Yes 38 ATTTAACATA TAAGCAAACA 20 20 Nucleic AcidSingle Linear Yes 39 CTGCCTGGTA CCCTGTTTTT 20 20 Nucleic Acid SingleLinear Yes 40 CTGCCTGGAA GGGTGTTTTT 20 20 Nucleic Acid Single Linear Yes41 CTGCCTGGTA CGGTGTTTTT 20 2510 Nucleic Acid Single Linear NO 42CGCCTCCCGG CCCCCTCCCC GCCCGACAGC GGCCGCTCGG GCCCCGGCTC 50 TCGGTTATAAGATGGCGGCG CTGAGCGGTG GCGGTGGTGG CGGCGCGGAG 100 CCGGGCCAGG CTCTGTTCAACGGGGACATG GAGCCCGAGG CCGGCGCCGG 150 CCGGCCCGCG GCCTCTTCGG CTGCGGACCCTGCCATTCCG GAGGAGGTGT 200 GGAATATCAA ACAAATGATT AAGTTGACAC AGGAACATATAGAGGCCCTA 250 TTGGACAAAT TTGGTGGGGA GCATAATCCA CCATCAATAT ATCTGGAGGC300 CTATGAAGAA TACACCAGCA AGCTAGATGC ACTCCAACAA AGAGAACAAC 350AGTTATTGGA ATCTCTGGGG AACGGAACTG ATTTTTCTGT TTCTAGCTCT 400 GCATCAATGGATACCGTTAC ATCTTCTTCC TCTTCTAGCC TTTCAGTGCT 450 ACCTTCATCT CTTTCAGTTTTTCAAAATCC CACAGATGTG GCACGGAGCA 500 ACCCCAAGTC ACCACAAAAA CCTATCGTTAGAGTCTTCCT GCCCAACAAA 550 CAGAGGACAG TGGTACCTGC AAGGTGTGGA GTTACAGTCCGAGACAGTCT 600 AAAGAAAGCA CTGATGATGA GAGGTCTAAT CCCAGAGTGC TGTGCTGTTT650 ACAGAATTCA GGATGGAGAG AAGAAACCAA TTGGTTGGGA CACTGATATT 700TCCTGGCTTA CTGGAGAAGA ATTGCATGTG GAAGTGTTGG AGAATGTTCC 750 ACTTACAACACACAACTTTG TACGAAAAAC GTTTTTCACC TTAGCATTTT 800 GTGACTTTTG TCGAAAGCTGCTTTTCCAGG GTTTCCGCTG TCAAACATGT 850 GGTTATAAAT TTCACCAGCG TTGTAGTACAGAAGTTCCAC TGATGTGTGT 900 TAATTATGAC CAACTTGATT TGCTGTTTGT CTCCAAGTTCTTTGAACACC 950 ACCCAATACC ACAGGAAGAG GCGTCCTTAG CAGAGACTGC CCTAACATCT1000 GGATCATCCC CTTCCGCACC CGCCTCGGAC TCTATTGGGC CCCAAATTCT 1050CACCAGTCCG TCTCCTTCAA AATCCATTCC AATTCCACAG CCCTTCCGAC 1100 CAGCAGATGAAGATCATCGA AATCAATTTG GGCAACGAGA CCGATCCTCA 1150 TCAGCTCCCA ATGTGCATATAAACACAATA GAACCTGTCA ATATTGATGA 1200 CTTGATTAGA GACCAAGGAT TTCGTGGTGATGGAGGATCA ACCACAGGTT 1250 TGTCTGCTAC CCCCCCTGCC TCATTACCTG GCTCACTAACTAACGTGAAA 1300 GCCTTACAGA AATCTCCAGG ACCTCAGCGA GAAAGGAAGT CATCTTCATC1350 CTCAGAAGAC AGGAATCGAA TGAAAACACT TGGTAGACGG GACTCGAGTG 1400ATGATTGGGA GATTCCTGAT GGGCAGATTA CAGTGGGACA AAGAATTGGA 1450 TCTGGATCATTTGGAACAGT CTACAAGGGA AAGTGGCATG GTGATGTGGC 1500 AGTGAAAATG TTGAATGTGACAGCACCTAC ACCTCAGCAG TTACAAGCCT 1550 TCAAAAATGA AGTAGGAGTA CTCAGGAAAACACGACATGT GAATATCCTA 1600 CTCTTCATGG GCTATTCCAC AAAGCCACAA CTGGCTATTGTTACCCAGTG 1650 GTGTGAGGGC TCCAGCTTGT ATCACCATCT CCATATCATT GAGACCAAAT1700 TTGAGATGAT CAAACTTATA GATATTGCAC GACAGACTGC ACAGGGCATG 1750GATTACTTAC ACGCCAAGTC AATCATCCAC AGAGACCTCA AGAGTAATAA 1800 TATATTTCTTCATGAAGACC TCACAGTAAA AATAGGTGAT TTTGGTCTAG 1850 CTACAGTGAA ATCTCGATGGAGTGGGTCCC ATCAGTTTGA ACAGTTGTCT 1900 GGATCCATTT TGTGGATGGC ACCAGAAGTCATCAGAATGC AAGATAAAAA 1950 TCCATACAGC TTTCAGTCAG ATGTATATGC ATTTGGGATTGTTCTGTATG 2000 AATTGATGAC TGGACAGTTA CCTTATTCAA ACATCAACAA CAGGGACCAG2050 ATAATTTTTA TGGTGGGACG AGGATACCTG TCTCCAGATC TCAGTAAGGT 2100ACGGAGTAAC TGTCCAAAAG CCATGAAGAG ATTAATGGCA GAGTGCCTCA 2150 AAAAGAAAAGAGATGAGAGA CCACTCTTTC CCCAAATTCT CGCCTCTATT 2200 GAGCTGCTGG CCCGCTCATTGCCAAAAATT CACCGCAGTG CATCAGAACC 2250 CTCCTTGAAT CGGGCTGGTT TCCAAACAGAGGATTTTAGT CTATATGCTT 2300 GTGCTTCTCC AAAAACACCC ATCCAGGCAG GGGGATATGGTGCGTTTCCT 2350 GTCCACTGAA ACAAATGAGT GAGAGAGTTC AGGAGAGTAG CAACAAAAGG2400 AAAATAAATG AACATATGTT TGCTTATATG TTAAATTGAA TAAAATACTC 2450TCTTTTTTTT TAAGGTGGAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2500 AAAAAAACCC2510

What is claimed is:
 1. An oligonucleotide 8 to 50 nucleotides in lengthwhich is targeted to a nucleic acid encoding human B-raf and whichinhibits the expression of human B-raf.
 2. The oligonucleotide of claim1 which is targeted to a translation initiation site, 3′ untranslatedregion, coding region or 5′ untranslated region of mRNA encoding humanB-raf.
 3. The oligonucleotide of claim 1 which has at least one modifiedbackbone linkage.
 4. The oligonucleotide of claim 1 wherein at least oneof the nucleotide units of the oligonucleotide is modified at the 2′position of the sugar moiety.
 5. The oligonucleotide of claim 1 in apharmaceutically acceptable carrier.
 6. The oligonucleotide of claim 1which is a chimeric oligonucleotide.
 7. The oligonucleotide of claim 1comprising SEQ ID NO: 3, 5, 7, 8, 9, 11, 13, 14, 17, 20, 21, 22, 32, 36,33, 31, 29 or
 23. 8. A method of inhibiting the expression of humanB-raf comprising contacting in vitro tissues or cells which expresshuman B-raf with an effective dose of the oligonucleotide of claim 1whereby expression of human B-raf is inhibited.
 9. The method of claim 8wherein the oligonucleotide is in a pharmaceutically acceptable carrier.10. The method of claim 8 wherein said expression of human raf isabnormal expression.
 11. A method of inhibiting hyperproliferation ofcells comprising contacting hyperproliferating cells in vitro with aneffective dose of the oligonucleotide of claim 1, wherebyhyperproliferation of cells is inhibited.